Isolation of Cyclic Protein-2 from Rat Seminiferous Tubule Fluid and Sertoli Cell Culture Medium1

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Abstract
Cyclic Protein-2 (CP-2), a stage-specific secretory product of the rat seminiferous epithelium, has been isolated from seminiferous tubule fluid (STF) and Sertoli cell culture medium. Isolation from STF was accomplished by mixing STF with radiolabeled proteins secreted by Stage VI-VII seminiferous tubules and sequential fractionation of these proteins by hydroxylapatite, DEAE-agarose, and quaternary amine ion-exchange chromatography. Radiolabeled proteins were used to identify the chromatographic fractions that contained CP-2. Through use of these procedures, a highly purified preparation of radioinert CP-2 was obtained from seminiferous tubule fluid. Cyclic Protein-2 was also isolated from Sertoli cell culture medium, indicating that the Sertoli cell is its most likely source. Preliminary characterization of CP-2 was conducted. First, CP-2 appeared to be highly enriched in methionine. Second, the molecular weight of CP-2 was found to be 20,000. Third, analysis by reverse-phase hydrophobic chromatography indicated that CP-2 was relatively hydrophobic. We conclude that CP-2 is a small hydrophobic glycoprotein secreted in vivo and in vitro in a stage-specific manner by Sertoli cells.